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The Journal of Immunology, 2005, 175: 6197-6204.
Copyright © 2005 by The American Association of Immunologists

CD80/CD86 Costimulation Regulates Acute Vascular Rejection1

Karoline A. Hosiawa2,*,{dagger}, Hao Wang2,{ddagger}, Mark E. DeVries*,{dagger}, Bertha Garcia§, Weihua Liu§, Dejun Zhou*, Ali Akram**,{dagger}{dagger}, Jifu Jiang{ddagger}, Hongtao Sun§, Mark J. Cameron, Robert Zhong3,{dagger},§,¶,|| and David J. Kelvin3

* Division of Experimental Therapeutics, University Health Network, Toronto General Research Institute, Toronto General Hospital, Toronto, Ontario, Canada; {dagger} Departments of Microbiology and Immunology, {ddagger} Department of Surgery, and § Department of Pathology, University of Western Ontario, London, Ontario, Canada; Multi-Organ Transplant Program, London Health Sciences Centre, London, Ontario, Canada; || Transplantation and Regeneration Group, Lawson Health Research Institute, London, Ontario, Canada; # Transplant Group, Robarts Research Institute, London, Ontario, Canada; ** Department of Immunology, University of Toronto, Toronto, Ontario, Canada; and {dagger}{dagger} Division of Infection, Injury, Immunity and Repair Research, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Xenotransplantation may provide the only solution to the shortage of human donor organs. Although hyperacute rejection associated with xenotransplantation can now be overcome, acute vascular rejection (AVR) remains a primary barrier to xenotransplantation. To date, standard immunosuppressive agents fail to block AVR or prolong xenograft survival. The present study was undertaken to determine the role of CD80/CD86 costimulatory molecules in regulating AVR. Lewis rat hearts were transplanted heterotopically into wild-type or IL-12, CD80- or CD86-deficient C57BL/6 mice. Wild-type recipients were treated with CD80 or CD86 neutralizing Ab with and without daily cyclosporin A (CsA, 15 mg/kg). Transplanted hearts in untreated wild-type recipients were rejected on postoperative days (POD) 17–21 and showed cell-mediated rejection (CMR) and AVR pathologies. In contrast, transplanted hearts in IL-12 and CD80 recipients or wild-type recipients treated with CD80 neutralizing Ab were rapidly rejected on POD 5 and 6 with AVR pathology. Interestingly, hearts transplanted into CD86 knockout recipients or wild-type recipients treated with CD86 neutralizing Ab underwent CMR on POD 17. Finally, blockade of CD86 but not CD80 rendered xenograft recipients sensitive to daily CsA therapy, leading to indefinite xenograft survival. To conclude, we demonstrate that AVR can be overcome by blocking the CD86 costimulatory pathway. Furthermore, we demonstrate that CD80 and CD86 have opposing roles in regulation of xenotransplantation rejection, where CD80 drives CMR and attenuates AVR while CD86 drives AVR. Most strikingly, indefinite xenograft survival can be achieved by suppressing AVR with CD86 neutralization in combination of CsA therapy, which inhibits CMR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Despite improvements in medical therapies, the outcome for patients with end-stage heart failure remains poor. Transplantation is the best therapy to "cure" these patients, however, fewer than 5% of those who need a cardiac transplant receive one in the United States each year (1). To solve the severe shortage of human heart donors, xenotransplantation may offer a great source to these patients who are desperately in need of an organ. In recent years considerable progress has been achieved in overcoming hyperacute rejection associated with xenotransplantation due to the generation of human complement regulatory protein transgenic pigs as well as pigs deficient in {alpha}1,3-galactosyltransferase (GalT)4 (2). Unfortunately, acute vascular rejection (AVR), an Ab-mediated disease, remains the primary barrier to successful xenotransplantation (3, 4). Furthermore conventional immunosuppressive drugs such as cyclosporin A (CsA), which is very effective in blocking cell-mediated rejection (CMR) in allotransplantation, have very limited effects on AVR in xenotransplantation. The future of xenotransplantation, therefore, depends on understanding the mechanisms of AVR and the identification of a novel approach to minimize/deviate AVR.

The underlying immune mechanisms driving AVR are poorly understood, although T cell-dependent and T cell-independent B cell responses as well as costimulatory molecules are likely involved in the initiation of the disease (5, 6, 7, 8, 9, 10, 11, 12, 13). CD86 (B7-2) and CD80 (B7-1) costimulatory molecules are part of an extensive family of costimulatory molecules critical for T cell activation (5, 14). CD86 and CD80 are expressed predominantly on APCs and bind CD28 expressed on T cells (7, 14). The higher affinity binding of CD86 to CD28 is critical for initiation of T cell responses, whereas the lower affinity binding of CD80 to CD28 is important for amplification of T cell responses (12, 15, 16, 17). Upon T cell activation, the preferential binding of CD80 to CTLA-4 results in inhibition of further T cell stimulation (18). It has been previously demonstrated that animals deficient in CD86 or CD80 do not have altered IgG isotype switching after immunization with adjuvant, although CD86-deficient mice show impaired isotype switching in the absence of adjuvant (19). Interestingly, CD80 transgenic mice exhibit suppression of T cell-dependent Ab responses (20). CD80 and CD86 have also been shown to differentially regulate B cell activation, wherein CD80 provides an inhibitory signal to B cell function and CD86 provides a stimulatory signal to B cell function (21). CD86 has also been shown to be important in initiation of IL-4 responses (22). Furthermore, CD80 and CD86 have been shown to have differential effects on the development of spontaneous autoimmune diabetes in NOD mice. Ab neutralization of CD80 was shown to exacerbate diabetes, whereas neutralization of CD86 was shown to abrogate the development of diabetes (23). Furthermore, disruption of CD28 signaling promoted the development of spontaneous autoimmune diabetes (24).

In light of these studies that suggest that CD80 and CD86 may influence divergent immunological responses, we examined the individual roles of CD80 and CD86 in regulating xenograft rejection in a concordant heterotopic cardiac rat-to-mouse transplantation model. We demonstrate that CD80 attenuates xenogeneic humoral responses and drives cellular responses, and CD86 drives xenogeneic humoral responses. Finally, we demonstrate that channeling xenogeneic immune responses through the CD80 costimulation pathway with a single dose of anti-CD86 Ab and daily CsA therapy results in indefinite xenograft survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Donors were 2-wk-old Lewis rats (25–30 g) purchased from Charles River Breeding Laboratories. C57BL/6, C57BL/6-CD80–/–, and C57BL/6-CD86–/– mice were purchased from The Jackson Laboratory, and C57BL/6-IL-12p40–/– mice were bred in pathogen-free conditions at the Robarts Research Institute (London, Ontario, Canada). After surgery, animals were housed in conventional housing at the Animal Care Veterinary Service facility, University of Western Ontario (London Ontario, Canada). Animals were cared for in accordance with the guidelines established by the Canadian Council on Animal Care. Recipients were treated with a single dose of 200 µg i.v. of CD80 (16-10A1) or CD86 (GL-1) neutralizing Abs (Bio Express) on postoperative day (POD) 0 or daily CsA (15 mg/kg, s.c.) until endpoint of rejection, where described.

Heterotopic heart transplantation

Food and water were not restricted for donors or recipients. Atropine and buprenorphine were routinely given s.c. before the induction of anesthesia by i.p. injection of pentobarbital. Intraabdominal heterotopic cardiac transplantation was performed as described (25). The beating of the graft was monitored daily by direct abdominal palpation. Grafts were considered to be at the endpoint when heartbeats were no longer palpable.

Graft histopathology

Hematoxylin-phloxine-saffron (HPS) stained microscopic sections were examined for severity of rejection by pathologists (B. Garcia and H. Sun) blinded to sample identity. Histological parameters including vasculitis, infarction, lymphocytic infiltration, thrombosis, hemorrhage, and fibrin (Martius scarlet blue stain) were all assigned scores on a scale from 0 to 4: 0, no change; 1, minimum change; 2, mild change; 3, moderate change; 4, severe change. Median scores of the six histological parameters (in the order listed) for cardiac xenografts were: C57BL/6-CD80–/– (POD 5) 0.8, 2.0, 0.2, 2.0, 2.0, 0; C57BL/6-CD86–/– (POD 6) 0.6, 0.0, 1.1, 0.0, 0.6, 0; C57BL/6-CD86–/– (POD 17) 1.3, 0.3, 1.2, 0.2, 0.9, 0; C57BL/6-IL-12p40–/– (POD 6) 3.0, 3.0, 0.0, 3.0, 2.0, 4.0; C57BL/6 (POD 6) 0.0, 1.0, 3.0, 0.0, 1.0, 0; C57BL/6 (POD 17–21) 1.0, 2.0, 3.0, 3.0, 3.0, 2.0.

Measurement of xenoreactive Abs

Lewis rat lymph node cells were isolated and incubated for 60 min at 4°C with sera from C57BL/6-CD80–/– (POD 5), C57BL/6-CD86–/– (POD 6 and 17), and C57BL/6 (POD 6 and 21) xenograft recipients at 1/25, 1/100, and 1/400 dilutions. Cells were then incubated for 60 min with goat anti-mouse PE-conjugated F(ab')2 IgM (Jackson ImmunoResearch Laboratories), or FITC-conjugated whole IgG1 or IgG2a (Caltag Laboratories). Flow cytometry acquisition was performed on a BD FACScan using CellQuest software (BD Biosciences).

Serum transfer

Sera were collected from anti-CD80- or anti-CD86 Ab-treated C57BL/6 mice on POD 5. Sera from over three transplanted mice were pooled and heat-inactivated at 56°C for 30 min. The 100 µl of serum was then injected (i.v.) on POD 0 into C57BL/6 mice. The beating of the graft was monitored daily by direct abdominal palpation.

Immunohistochemistry

Sections (4 µm) were cut from frozen tissues and fixed in 100% acetone for 10 min. Sections were stained using goat anti-mouse IgM or IgG-biotin (Caltag Laboratories), CD4-biotin (YTS 191.1.2; Cedarlane Laboratories), CD8-biotin (53-6.7; BD Biosciences), or biotin-rat IgG2a or IgG2b (Cedarlane Laboratories). The negative control for IgG and IgM staining was PBS alone. Sections were stained by an Elite Vectastain ABC kit (Vector Laboratories) and followed by the 3,3'-diaminobenzidine substrate (BD Biosciences). IgM and IgG staining was quantified as number of positive cells/x20 field.

Real-time PCR

Total RNA was purified from tissues using TriPure Reagent (Roche) according to the manufacturer’s specifications. Genomic DNA was removed using a RNase-free DNase on-column treatment (Qiagen). mRNA (500 µg) was reverse transcribed into cDNA using SuperScript II (Invitrogen Life Technologies). cDNA (0.25 µl) was amplified with the SYBR Green Master Mix (Applied Biosystems) using the ABI 7900 Sequence Detection System (Applied Biosystems) using the following primers: murine granzyme B (sense) 5'-AAGCTGAAGAGTAAGGCCAAGAG-3', (antisense) 5'-CAACAAGCCACATAGCACACA-3'; murine IL-12p40 (sense) 5'-CCATTGAAC TGGCGTTGGA-3', (antisense) 5'-CTGGTTTGATGATGTCCCTGA-3'; murine IL-12R{beta}1 (sense) 5'-ATGGCTGCTGCGTTGAGAA-3', (antisense) 5'-AGCACTCATAGTCT GTCTTGGA-3', murine Fas ligand (FasL, sense) 5'-TCTGTGGCTACCGGTGGTATT-3', (antisense) 5'-TGGAAGAGCTGATAC ATTCCTAATCC-3'; murine 18 S (sense) 5'-TCGGAACTGAGGCCATGATT-3', (antisense) 5'-TTTCGCTCTGGTCCGTCTTG-3'; and IFN-{gamma} (sense) 5'-TGCATTCATGAGTATTGCCAAGT-3', (antisense) 5'-GCTGGATTCCGGCAACAG-3'. The relative expression of each gene was normalized to 18 S rRNA control. The values represent mean mRNA expression ± SD.

Statistics

We used the two-tailed unpaired Student’s t test for comparison between two groups. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD80 deficiency results in aggressive Ab-mediated rejection whereas CD86 deficiency results in CMR

As we have reported previously, C57BL/6 wild-type (WT) recipients had an early infiltration of lymphocytes at POD 6, but did not reject rat hearts until POD 17–21 (26). At endpoint, defined as loss of detectable heartbeat, transplanted rat hearts in WT recipients had a mixed pathology with characteristics of both Ab-mediated AVR thrombosis formation, hemorrhage and fibrin deposition, and the characteristics of CMR, namely lymphocyte infiltration (Fig. 1A and Table I). Remarkably, xenograft hearts in CD80–/– recipients were rejected on POD 5 and were dominated by AVR pathology with thrombosis formation, hemorrhage, and deceased cellular infiltration (Fig. 1A and Table I). In contrast to CD80–/– recipients, CD86–/– recipients rejected rat hearts on POD 17. Histological examination showed a typical feature of CMR, characterized by a heavy infiltration of lymphocytes with "clean" vessels (Fig. 1A and Table I). To determine whether CD80 and CD86 regulated Ab-mediated rejection or CMR, we next treated WT recipients with neutralizing Abs to CD80 or CD86. This treatment could determine whether the intrinsic immune response in WT mice could be channeled toward either Ab-mediated AVR or CMR through therapeutic intervention at the time of xenotransplantation. Similar to the results using CD80–/– recipients, treatment of WT recipients with a single dose of CD80-neutralizing Ab (200 µg) resulted in rejection of xenograft hearts on POD 6 with Ab-mediated rejection (AVR) pathology (Table I). Treatment of WT recipients with a single dose of CD86-neutralizing Ab (200 µg) resulted in rejection of xenograft hearts on POD 14 with a pathology characteristic of CMR (Table I), further supporting the data from CD86–/– recipients. Even though grafts rejected in anti-CD86-treated WT recipients and CD86–/– recipients were dominated by CMR, some low-grade hemorrhage was observed. To investigate the role of Abs in xenograft rejection, we measured serum and intragraft levels of anti-rat IgM and IgG Abs.



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  		FIGURE 1. Onset of Ab-driven AVR at POD 5 in the absence of CD80 costimulation and onset of CMR at POD 17 in the absence of CD86 costimulation. A, Pathology of heart xenografts. HPS staining of xenograft hearts in CD80–/– (POD 5), CD86–/– (POD 6 and 17), and WT (POD 6 and 21) recipients. Scale bar, 100 µm. Arrows indicate thrombosis and arrowheads hemorrhage. B–D, Quantitation of serum xenoreactive Abs. Serum mouse anti-rat IgM (B), IgG1 (C), and IgG2a (D) Abs were measured in CD80–/– (POD 0 and 5), CD86–/– (POD 0, 6, and 17), and WT (POD 0, 6, and 21) recipients by flow cytometry. Data represent mean fluorescent intensity ± SD (n = 4). *, p < 0.05 vs CD86–/– POD 6, **, p < 0.01 vs CD86–/– POD 6. POD 5 is also the endpoint of rejection in CD80–/– mice.

 

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  		Table I. Mean xenograft survival time, treatment and pathologya

 
Elevation of xenogeneic anti-rat IgG1 and IgG2a Abs correlate with aggressive Ab-mediated rejection

To evaluate whether AVR is driven by a xenogeneic Ab response, we measured serum levels of anti-rat Abs of the IgM and IgG isotype and intragraft IgM and IgG deposition. Both serum levels of anti-rat IgM Ab and intragraft IgM deposition were similar between WT, CD80–/–, and CD86–/– recipients on POD 5 and 6 (Figs. 1B and 2A). That finding indicates that increases in IgM xenoantibodies, over baseline levels (POD 0), do not correlate with AVR. In contrast, CD80–/– recipients had increased serumanti-rat IgG1 and IgG2a levels when rat hearts were rejected on POD 5 compared with WT and CD86–/– recipients on POD 6 (Fig. 1, C and D). Levels of anti-rat IgG1 and IgG2a Abs increased at the time of rejection on POD 17–21 in WT and CD86–/– recipients over baseline levels on POD 0 (Fig. 1, C and D). Titration of anti-rat IgG and IgM isotypes over 1/25, 1/100, and 1/400 serum dilutions showed similar results (data not shown). High levels of circulating serum anti-rat IgG1 and IgG2a Abs correlate with Ab-mediated AVR, indicating that CD80 has a negative effect on IgG xenoantibody generation and onset of AVR. Similarly to serum Ab levels, intragraft IgG Ab deposition was significantly increased at POD 5 in CD80–/– recipients compared with levels in CD86–/– recipients on POD 6 (Fig. 2B). The levels of deposited IgG also increased at time of CMR in CD86–/– recipients. These data suggest that CD80 negatively regulates the generation of anti-rat IgG Abs and attenuates Ab-driven AVR, whereas CD86 appears to promote or be permissive to the generation of anti-rat IgG Abs and is critical for the early onset of Ab-mediated AVR. We examined whether anti-rat Abs cause xenograft rejection by transferring serum containing high or low levels of anti-rat Abs to WT mice before transplantation.



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  		FIGURE 2. Intragraft IgM and IgG deposition. Enumeration of intragraft IgM (A), and IgG (B) deposition in CD80–/– (POD 5), CD86–/– (POD 6 and 17), and WT (POD 6 and 21) grafts as measured by immunohistochemical staining (n = 6). IgM and IgG deposition shown as number of positive cells/x20 field ± SD. **, p < 0.01 vs CD86–/– POD 6. {ddagger}, POD 5 is also the endpoint of rejection in CD80–/– mice.

 
Serum from anti-CD80-treated mice transfers aggressive Ab-mediated rejection

To assess whether elevated anti-rat Abs are a cause of graft rejection we tested whether Abs in sera can transfer rejection. We transferred heat-inactivated sera pooled from anti-CD80 Ab-treated WT recipients collected on POD 5 into WT recipients before transplantation. Alternatively, we transferred heat-inactivated sera pooled from anti-CD86 Ab-treated WT recipients collected on POD 5 into WT recipients before transplantation. As demonstrated in Fig. 3, transfer of serum collected from anti-CD80 Ab-treated mice accelerated xenograft rejection from POD 17 to 5. Meanwhile, transfer of serum collected from anti-CD86 Ab-treated mice only accelerated xenograft rejection from POD 17 to 15. This suggests that Abs play a critical role in xenograft rejection. Due to the importance of T cells in B cell responses, we evaluated the role of CD80 and CD86 in IL-12 expression and T cell infiltration during xenograft rejection.



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  		FIGURE 3. Transfer of serum collected from anti-CD80 Ab-treated mice accelerates AVR in C57BL/6 mice. Sera were pooled from >3 anti-CD80 or anti-CD86 Ab-treated WT mice on POD 5. Pooled serum was heat-inactivated and injected i.v. (100 µl) into WT mice on POD 0 (n = 4). Data are represented as a percentage of survival in untreated WT mice and WT mice injected with pooled serum from anti-CD80 or anti-CD86 Ab-treated mice.

 
Diminished IL-12 expression and T cell infiltration accompanies the onset of aggressive Ab-mediated rejection pathology in the absence of CD80

To determine the effect of CD80 and CD86 costimulatory molecules on induction of Th1 responses and T cell recruitment, we measured the levels of IL-12 expression and T cell infiltration into xenograft hearts. We previously reported that IL-12 attenuates Ab-driven AVR (26). Interestingly, both CD80–/– and IL-12p40–/– recipients rejected xenograft hearts on POD 5 and 6 with an AVR pathology (Table I). To determine whether the Ab responses observed in CD80–/– recipients correlated with an impaired IL-12 response, we measured intragraft and splenic IL-12p40 mRNA expression by real-time PCR. At the time of rejection, CD80–/– recipients showed a significant decrease in expression of intragraft IL-12p40 mRNA compared with WT or CD86–/– recipients on POD 5 and 6 (Fig. 4A). CD86–/– recipients also showed high intragraft IL-12p40 mRNA expression on POD 17/endpoint. These data demonstrate that CD80–/– and CD86–/– recipients differentially regulate IL-12p40 expression, indicating that CD80 promotes IL-12p40 expression, whereas CD86 suppresses IL-12p40 expression. Similarly, CD80–/– recipients showed decreased mRNA expression of IL-12p40 and IL-12R{beta}1 in the spleens on POD 5, compared with CD86–/– recipients on POD 6 (Fig. 4, B and C). Because IL-12 is critical for Th1 differentiation and Th1-like responses are associated with CMR, we characterized the presence of T cells in transplanted hearts to determine whether they in fact contribute to Ab-mediated or xenograft CMR. As we previously reported (26), WT recipients had extensive T cell (CD4+ and CD8+) intragraft infiltration on POD 6, which declined at the time of rejection (POD 17–21). CD86–/– recipients showed significantly higher numbers of graft infiltrating CD4+ T cells on both POD 6 and 17/endpoint and significantly higher numbers of graft infiltrating CD8+ T cells on POD 17/endpoint compared with grafts from CD80–/– recipients (Fig. 4D). These results indicate that although rejection of rat hearts in WT recipients is due to both xenogeneic anti-rat humoral responses and prior T cell infiltration, rejection in CD86–/– recipients appears to be T cell dependent. To further resolve the involvement of T cells in xenograft rejection, we measured the expression of T cell cytokines and cytotoxic effector molecules.



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  		FIGURE 4. Reduced expression of intragraft and splenic IL-12p40 mRNA at endpoint of rejection (AVR) on POD 5 correlates with absence of graft-infiltrating CD4+ and CD8+ T cells. Quantitation of intragraft and splenic IL-12p40 mRNA expression. Relative IL-12p40 mRNA expression ± SD was quantified in grafts (A) from CD80–/– (POD 5), CD86–/– (POD 6 and 17), and WT (POD 6 and 21) recipients by real-time PCR (n = 3). Relative IL-12p40 (B) and IL-12R{beta}1 (C) mRNA expression ± SD was quantified in spleens from CD80–/– (POD 5), CD86–/– (POD 6), and WT (POD 6) recipients by real-time PCR (n = 3). D, Enumeration of graft-infiltrating CD4+ and CD8+ cells. Immunohistochemical staining of CD80–/– (POD 5) and CD86–/– (POD 6 and 17) grafts with Abs against CD4 or CD8. Data represent mean number of cells/x20 field ± SD (n = 6). *, p < 0.05 vs CD86–/– POD 6. **, p < 0.01 vs CD86–/– POD 6. {ddagger}, POD 5 is also the endpoint of rejection in CD80–/– mice.

 
Persisting IFN-{gamma} and T cell effector molecule expression correlates with the onset of cell-mediated pathology in absence of CD86

To delineate the mechanism of CMR in the absence of CD86, we measured the expression of cytokines and cytotoxic effector molecules. We tested the expression of IFN-{gamma} and mediators of T cell cytotoxicity in the xenograft. We demonstrate that CD86–/– recipients express high levels of IFN-{gamma}, granzyme B, and FasL mRNA, similar to WT recipients (Fig. 5A-C). Collectively, these data suggest that CD80 and CD86 costimulatory molecules modulate distinct T cell xenotransplantation responses. CD80, but not CD86, appears to promote T cell infiltration and T cell cytotoxicity accompanied by IL-12p40 and IFN-{gamma} expression. Furthermore, evidence of cellular infiltration and lack of anti-donor Ab responses in CD86–/– or CD86 neutralized recipients transplanted with Lewis rat hearts is similar in many respects to the pathology observed in rejected grafts from allotransplanted mice. Based on the efficacy of standard immunosuppression on inhibition of T cell responses, we investigated whether CMR can be suppressed with CsA.



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  		FIGURE 5. Intragraft IFN-{gamma}, granzyme B, and FasL mRNA expression. Relative intragraft mRNA expression ± SD of murine IFN-{gamma} (A), murine granzyme B (B), and murine FasL (C) in CD80–/– (POD 5), CD86–/– (POD 6 and 17), and WT (POD 6 and 21) recipients quantified by real-time PCR. *, p < 0.05 and **, p < 0.01 vs CD86–/– POD 6. {ddagger}, POD 5 is also the endpoint of rejection in CD80–/– mice.

 
CsA treatment leads to indefinite xenograft survival in CD86-deficient, but not CD80-deficient mice

Acute allograft rejection in mice can be readily controlled with conventional immunosuppression, however conventional immunosuppression alone is ineffective in suppressing xenograft rejection (27, 28). We sought to determine whether altering the mechanism of xenograft rejection from CD86-driven Ab-mediated rejection to CD80-driven CMR would make recipients sensitive to CsA immunosuppression. This was accomplished by treating WT recipients with CD86-neutralizing Ab. We hypothesized that the CMR pathology associated with the CD80 pathway, like in allotransplantation, could be controlled with CsA. WT mice were treated with a single dose of CD86- or CD80-neutralizing mAb (200 µg) combined with daily CsA or daily CsA alone. In contrast to CsA treatment alone, rat hearts transplanted into WT mice treated with CD86-neutralizing mAb plus CsA survived beyond 100 days with no sign of rejection in 100% of recipient mice (Fig. 6, A and B). We also found that rat heart transplants in CD86–/– recipients treated with daily CsA had dramatically increased survival, with 29% of the grafts surviving beyond 100 days (Fig. 6B). Treatment with CD80 neutralizing mAb combined with daily CsA, however, only resulted in graft survival of 14 ± 3 days (Fig. 6B). The difference between the numbers of xenografts surviving beyond 100 days with CD86 Ab neutralization in WT recipients and in CD86-deficient animals may be due to compensatory immunological mechanisms in CD86–/– mice.



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  		FIGURE 6. CsA therapy combined with CD86 neutralization leads to indefinite xenograft survival in WT recipients. A, Graft pathology in WT recipients treated with CsA and a single dose of CD80- or CD86-neutralizing Abs. HPS stained grafts in WT recipients treated with CsA alone (n = 6) shown on POD 27/endpoint, WT recipients treated with CsA and CD80-neutralizing Ab (n = 6) shown on POD 14/endpoint, and WT recipients treated with CsA and CD86-neutralizing Ab (n = 6) shown on POD 100 (time of sacrifice) with no sign of rejection. Arrows indicate thrombosis and arrowheads hemorrhage. Scale bar, 100 µm. B, Percentage of xenograft survival in WT recipients treated with CsA alone, CsA and CD80-neutralizing Ab, or CsA and CD86-neutralizing Ab. Percentage of xenograft survival in CD80–/– (n = 6) and CD86–/– (n = 14) recipients treated daily with CsA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study we demonstrate that C57BL/6 WT xenograft recipients develop CMR on POD 6, and then succumb to Ab-mediated rejection and CMR on POD 17–21 and show elevated intragraft and splenic IL-12p40 expression on POD 6 but not at POD 17–21. WT recipients were resistant to CsA therapy. In contrast, in the absence of CD80, xenograft recipients shifted to aggressive Ab-mediated rejection on POD 5, at which time they show elevated xenogeneic IgG Ab responses, lack T cell infiltration to the graft, and show decreased intragraft and splenic IL-12p40 expression. In the absence of CD80, recipients are unresponsive to CsA therapy. On the contrary, in the absence of CD86, recipients reject xenograft hearts with the same kinetics as WT recipients, however the pathology is strikingly different. In the absence of CD86, xenograft recipients succumb to CMR on POD 17, show decreased xenogeneic IgG Ab responses, show sustained T cell infiltration to the graft on both POD 6 and 17, and show sustained intragraft and splenic IL-12p40 expression on both POD 6 and 17. In the absence of CD86, recipients are responsive to CsA, leading to indefinite xenograft survival. These data demonstrate that CD80 and CD86 have mechanistically divergent roles in regulating xenogeneic immune responses. The present studies also demonstrate that anti-rat Abs cause accelerated xenograft rejection. We demonstrate that CD80 drives cell-mediated xenogeneic responses and attenuates xenogeneic humoral responses, whereas CD86 drives humoral xenogeneic responses. Most importantly, we demonstrate that channeling xenogeneic immune responses toward the CD80 pathway through CD86 neutralization renders the recipient sensitive to CsA therapy. Most remarkably, a single dose of CD86-neutralizing Ab followed by daily CsA therapy increased graft survival to beyond 100 days in 100% of cases. This was in contrast to the below 100% compliance observed in CD80 and CD86 knockout mice. This difference between Ab neutralization of CD86 and using CD86-deficient mice may be attributed to compensatory molecules in knockout mice. Although many other costimulatory molecules such as inducible costimulator, 4-1BB, and PD-1 are involved in T and B cell responses, the dramatic and opposing roles for CD80 and CD86 costimulatory molecules uncovered in our present study suggest that CD80 and CD86 play a major role in regulation of Ab-mediated AVR. This is demonstrated by dramatically altering the outcome of xenotransplantation with manipulating either CD80 or CD86 costimulatory molecules alone, while other costimulatory pathways such as CD40/CD40L, inducible costimulator or 4-1BB are left intact.

The role of CD80 and CD86 in allotransplantation has been studied extensively. Neutralization of CD86 in allotransplantation significantly prolongs heart allograft survival by 1–3 wk (16, 29, 30), whereas neutralization of CD80 has little or no effect (16, 29). Previous studies have demonstrated that blockade of both murine CD80 and CD86 costimulatory molecules increases cardiac xenograft survival but does not extend it indefinitely (31). This result indicates that other costimulatory pathways may regulate late xenogeneic immune responses, whereas CD80 and CD86 regulate early xenogeneic immune responses. However, it has been demonstrated that blocking of both rat CD80 and CD86 costimulatory molecules does not affect survival of rat cardiac xenografts in mice recipients (31). This suggests that xenograft Ags are processed predominantly by the indirect Ag recognition pathway, independently of donor APCs and their costimulatory molecules. Hence, although it has been reported that porcine CD80/CD86 and CD40 costimulatory molecules are capable of interacting with their cognate receptors on human CD4+ cells, resulting in proliferative responses (32), the donor molecules may not have a role in xenograft survival.

The underlying mechanism for how CD80 and CD86 costimulatory molecules regulate xenogeneic immune responses is likely due to regulation of cytokine cascades as IL-12 is differentially regulated between mice deficient in CD80 or CD86. As previously demonstrated, IL-12 expression is observed in "waves" posttransplantation, as seen in low levels of expression at the time or rejection compared with earlier time points. Our data suggests that IL-12 is associated with CMR and inversely correlated with Ab-mediated rejection. It has been demonstrated that IL-12 sustains CD40L (CD154) expression on activated T cells and prolonged CD40L expression has been shown to suppress terminal B cell differentiation (33). This may explain why CD86–/–, but not CD80–/–, recipients show low xenogeneic B cell responses and are sensitive to CsA therapy. Alternatively, IL-12 may regulate B cell responses directly, as it has been shown that B cells express the IL-12R and are able to respond to IL-12, in addition to secreting IL-12 under certain conditions (34, 35, 36). Hence differential regulation of IL-12p40 by CD80 and CD86 may result in opposing effects on B cell responses. Furthermore, we demonstrate that transfer of heat-inactivated serum, collected from anti-CD80 Ab-treated WT mice, which contains high levels of anti-rat IgM and IgG Abs, but no active complement, accelerates xenograft rejection from POD 17 to 5. In contrast, transfer of heat-inactivated serum, collected from anti-CD86 Ab treated WT mice, which contains low levels of anti-rat IgM and IgG Abs does not accelerate xenograft rejection. These data suggest that Abs are a cause, and not an effect, of xenograft rejection. Overcoming Ab-mediated xenograft rejection presents as the main barrier to clinical xenotransplantation. Generation of the GalT knockout pigs has been a major breakthrough (2). Deletion of GalT in donor pig hearts has been demonstrated to alleviate hyperacute rejection in nonhuman primate recipients, although the xenografts are still rejected 2–6 mo posttransplant (37). Although reaching 6 mo xenograft survival has exceeded previous attempts at pig-to-nonhuman primate transplantation, deletion of GalT does not however overcome the subsequent Ab-mediated rejection. Nonhuman primate recipients of GalT pig hearts, as well as recipients of life supporting GalT renal xenografts, still demonstrate the development of thrombotic microangiopathy. Thrombotic microangiopathy may be a result of endothelial cell activation by low levels of non-Gal{alpha}1,3Gal directed Abs that encourage fibrin and platelet adhesion (37, 38). Furthermore, heavy immunosuppressive protocols were used in these studies, resulting in high mortality in recipients. Hence, to establish long-term xenograft survival we need to address the mechanisms driving xenogeneic Ab responses as well as the ensuing cellular responses.

Xenogeneic cellular responses are often overlooked because of the sudden onset on Ab-mediated rejection, resulting in early graft loss. Little is understood about the mechanisms of cell-mediated xenograft rejection. CD4+ T cells have been shown to be critical in mediating cellular graft injury, but their regulation by the immune system is unclear. Xenogeneic graft rejection has been thought to be similar to allogeneic cellular rejection in strength and specificity. Both allogeneic and xenogeneic cellular rejection is thought to involve the TCR repertoire, T cell accessory molecule interactions, and cytokine production (39). One particular difference between allogeneic and xenogeneic cellular responses is the Ag recognition pathways. Allogeneic Ags are thought to be processed through both the direct and indirect Ag recognition, whereas xenogeneic Ags are thought to be processed predominantly through the direct Ag recognition, not involving donor APCs (40). This may be responsible for the differential regulation of allogeneic and xenogeneic cell-mediated responses.

Our results demonstrate that targeting recipient CD86 may shift the mechanism of rejection from Ab-mediated to cell-mediated, providing a novel therapy to future preclinical and clinical xenotransplantation. Because xenogeneic CMR is sensitive to CsA therapy, a novel therapy may easily be applied to larger animal nonhuman primate models of xenotransplantation. Anti-CD80/CD86 Ab therapy and CsA have already demonstrated efficacy in suppressing nonhuman primate and human immune responses (41). Shifting the immune response from CD86 to CD80, by neutralization of CD86, may alter the number of CD4+CD25+ T regulatory cells posttransplant, which render the animal sensitive to CsA treatment, but this remains to be explored. In addition to the xenotransplantation models, the finding that neutralization of CD86 combined with CsA therapy leads to indefinite xenograft survival has significant implications for allotransplantation. There is significant graft loss due to allogeneic Ab-mediated acute rejection in 5–15% of human transplant recipients. Allogeneic Ab-mediated rejection differs from xenogeneic Ab-mediated rejection by the source of the antigenic determinant. Human and nonhuman primate xenograft recipients react to the Gal{alpha}1,3Gal epitope on pig organs as well as other unknown non-Gal{alpha}1,3Gal epitopes (42). In contrast, in allotransplantation, patients who have been in contact with HLA or ABO alloantigens through transfusions, pregnancies and previous transplantation are predisposed to Ab-mediated allograft rejection (40). Generation of allogeneic Ab responses leads to complement activation, ischemia-reperfusion injury, and activation of Ab-dependent cell cytotoxicity by NK cells and macrophages, leading to early allograft rejection (40). Allogeneic Ab-mediated rejection is not responsive to steroid and anti-lymphocyte therapy (43). Although the Ab responses in acute allograft rejection and AVR of xenografts are directed at different Ags, both are a rapid B cell response. Therefore, neutralization of CD86 combined with CsA therapy may be successful in treating cases of acute allograft rejection.

In conclusion, we demonstrate that CD80 and CD86 costimulatory molecules have opposing effects on xenogeneic responses. We show that a one-time dose of CD86-neutralizing Ab suppresses xenogeneic B cell responses and AVR and renders xenograft recipients sensitive to CsA therapy.


   Acknowledgments
 
We thank C. Cameron and S. Bosinger for critical review of the manuscript, C. Meagher for thoughtful advice during the course of these studies, L. Xu for technical assistance, and M. Sequeira for administrative assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Genome Canada, Canadian Institutes of Health Research, Health Research Programs of Excellence (Transnet), Canadian Institutes of Health Research (operating grants to D.J.K. and R.Z.), and the Ontario Research and Development Challenge Fund. Back

2 K.A.H. and H.W. contributed equally to this work. Back

3 Address correspondence and reprint requests Dr. David J. Kelvin, Division of Experimental Therapeutics, University Health Network, 200 Elizabeth Street, Max Bell Research Centre, Fifth Floor, Room 425, Toronto M5G 2C4, Ontario, Canada; E-mail address: dkelvin{at}uhnres.utoronto.ca or Dr. Robert Zhong, London Health Sciences Centre, Room 9-OF9, University Campus, 339 Windermere Road, London N6A 5A5, Ontario, Canada; E-mail address: zzhong{at}uwo.ca Back

4 Abbreviations used in this paper: GalT, {alpha}1,3-galactosyltransferase; AVR, acute vascular rejection; CMR, cell-mediated rejection; FasL, Fas ligand; CsA, cyclosporin A; HPS, hematoxylin-phloxine-saffron; POD, postoperative day; WT, wild type. Back

Received for publication March 30, 2005. Accepted for publication August 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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